1-Origin of Dendrimers:

Polymer chemistry and technology have traditionally focused on linear polymers, which are widely in use. Linear macromolecules only occasionally contain some smaller or longer branches. In the recent past it has been found that the properties of highly branched macromolecules can be very different from conventional polymers. The structure of these materials has also a great impact on their applications. First discovered in the early 1980’s by Donald Tomalia and co-workers, these hyper branched molecules were called dendrimers. The term originates from ‘dendron’ meaning a tree in Greek. At the same time, Newkome’s group independently reported synthesis of similar macromolecules. They called them arborols from the Latin word ‘arbor’ also meaning a tree. The term cascade molecule is also used, but ‘dendrimer’ is the best established one.Dendrimer chemistry began nearly two decades ago with the synthesis and characterization of a family of highly branched macromolecules.( Gorman and Smith)( Gorman

et al)The general Dendrimer structure consisted of an inner core molecule with hyper branched polymeric structures, known as dendrons, extending outward from the core. Each dendritic branch was terminated with a chosen periphery structure , (Fig. 1) The ability to synthesize well-defined monodisperse macromolecules using repetitive activate/couple growth cycles added a new dimension to polymer chemistry.At the nanoscale (molecular level),there are relatively few natural examples of this architecture. Most notable are glycogen and amylopectin, macromolecular hyper branched structures that nature uses for energy storage.( Donald A. Tomalia)

The complexity of organic synthesis has been steadily enhanced by utilizing the known hybridization states of carbon and specific heteroatom to produce key molecular-level hydrocarbon building blocks (modules) and functional groups (connectors),These two construction parameters have been used to assemble literally millions of more complex structures, Relatively small (i.e. !1 nm) molecules were produced, the structures of which could be controlled as a function of their shape, mass, flexibility, and functional group placement, Based on the various hybridization states of carbon, at least four major carboskeletal architectures are known (Corey,

Cheng ) (Heilbronner, Dunitz) They are recognized as the(I) linear, (II) bridged (2D/3D), (III) branched and , more recently, the (IV) dendritic (cascade) (Buhleier et al) (Fig.2).Numerous synthetic strategies have been reported for the preparation of these materials, which have led to a broad range of dendritic structures. Presently, this architectural class consists of four dendritic (cascade) subclasses: (a) randomhyper branched polymers, (b) dendrigraft polymers, (c) dendrons, and (d) Dendrimers (Fig. 3).The order of this subset, from (a) to (d), reflects the relative degree of structural control present in each of these dendritic architectures ( Tomalia et al).All dendritic polymers are open, covalent assemblies of branch cells (Fig. 3a). They may be organized as very symmetrical, monodispersed arrays, as is the case for Dendrimers, or as irregular, polydispersed assemblies that typically define random, hyper branched polymers.The dendritic structure is characterized by ‘layers’ between each focal point (or cascade) called generations (shown as circles on Fig.4) ( Supattapone et al). The exact numbering of generations has been the subject of some confusion , the dendrimer generation is defined as the number of focal points (cascade points) when going from

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the core to the surface, a generation 5 (G5) dendrimer thus has 5 cascade points between the core and the surface.The core is sometimes denoted generation ‘zero’ (G0), as no cascade points are present, for a polypropylene imine (PPI) dendrimer, the core is 1,4-diaminobutane which has no cascade points, for a polyamido amine (PAMAM) ‘Starburst™’ dendrimer the core is ammonia etc. (hydrogen substituents are not considereda focal point). In PAMAM dendrimers the intermediate compounds having carboxylate surface groups are denoted half-generation dendrimers, that is dendrimers of e.g. G1.5 or G2.5.The dendrimer design can be based on a large variety of linkages, such as polyamines (PPI dendrimers), a mix of polyamides and amines (PAMAM dendrimers) or built up by more hydrophobic poly(aryl ether) subunits (C. J. Hawker et al). More recent examples are dendrimer designs based on carbohydrate or calixarene core structures, or containing ‘third period’ elements like silicon or phosphorus (J. P. Majoral and A. M. Caminade), just to give a few examples (Fig.5).

2-Dendrimer synthesis:

In contrast to traditional polymers, dendrimers are unique core–shell structures possessing three basic architectural components : (I) a core, (II) an interior of shells (generations) consisting of repeating branch-cell units, and (III) terminal functional groups (the outer shell or periphery) ( Donald A. Tomalia).

2.1-"Divergent" Dendrimer Growth:

The development of new synthetic schemes enabled manipulation of the structure of dendrimers. One scheme, the divergent strategy, In the divergent methods, dendrimer grows outwards from a multifunctional core molecule.The core molecule reacts with monomer molecules containing one reactive and two dormant groups giving the first generation dendrimer. Then the new periphery of the molecule is activated for reactions with more monomers. The process is repeated for several generations and a dendrimer is built layer after layer (Fig.6). The divergent approach is successful for the production of large quantities of dendrimers. Problemsoccur from side reactions and incomplete reactions of the end groups that lead to structure defects. To prevent side reactions and to force reactions to completion large excess of reagents is required. It causes some difficulties in the purification of the final product.

2.2-"Convergent" Dendrimer Growth: To eliminate this problem, a convergent strategy for the synthesis of dendrimers was developed. This strategy originates with the peripheral units and uses the same iterative deprotection and coupling scheme to construct the dendritic branches inward (Fig.7).Each coupling cycle designates a new layer of branch points referred to as a generation. The dendritic arms are ultimately terminated with a linking unit that is used for core attachment. The convergent growth method has several advantages. Itis relatively easy to purify the desired product and the occurrence of defects in the final structure is minimized.

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Monodispersity becomes a problem at high molecular weights therefore, the convergent strategy is not preferred when synthesizing large generation dendrimers.

2.3-"Hypercores" and "Branched Monomers":

In the early 1990s, the area of synthetic methodology in dendrimer research was led by the Fréchet group at Cornell University. After the development of the convergent approach, their efforts focused on the acceleration of dendrimer syntheses. The outcome of this research was the demonstration (Fig. 8) of 'hypercores' and 'branched monomers'. These methods involve the pre-assembly of oligomeric species which can then be linked together to give dendrimers in fewer steps or higher yields. Hypercores and branched monomers allow the chemist to devise synthetic strategies that are more convergent in the classical synthetic sense of the word. ( AN Shipway)

2.4-"Double Exponential" and "Mixed" Growth:

The most recent fundamental breakthrough in the practice of dendrimer synthesis has come with the concept and implications of 'double exponential' growth (Fig. 9). Double exponential growth, similar to a rapid growth technique for linear polymers, involves an AB2 monomer with orthogonal protecting groups for the A and B functionalities. This approach allows the preparation of monomers for both convergent and divergent growth from a single starting material. These two products are reacted together to give an orthogonally protected trimer, which may be used to repeat the growth process again. The strength of double exponential growth is more subtle than the ability to build large dendrimers in relatively few steps. In fact, double exponential growth is so fast that it can be repeated only two or perhaps three times before further growth becomes impossible. The double exponential methodology provides a means whereby a dendritic fragment can be extended in either the convergent or the divergent direction as required. In this way, the positive aspects of both approaches can be accessed without the necessity to bow to their shortcomings. ( AN Shipway)

3-MOLECULAR STRUCTURE:

The dendrimer structure can be divided into three parts:–The multivalent surface, with a high number of potentialreactive sites.–The ‘outer shell’ just beneath the surface having a well-definedmicroenvironment protected from the outside by the dendrimersurface.–The core, which in higher generation dendrimers is protectedfrom the surroundings, creating a microenvironment surrounded by the dendritic branches (C. J. Hawker et al).These three architectural components (core, interior, and periphery) essentially determine the physical and chemical properties, as well as the overall size, shape, and flexibility of a dendrimer. It is important to note that dendrimer diameters increase linearly as a function of shells or generations added, whereas the terminal functional groups increase exponentially as a function of generation.

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As a consequence, lower generations are generally open, floppy structures, whereas higher generations become robust, less deformable spheroids, ellipsoids, or cylinders depending on the shape and directionality of the core (Donald A. Tomalia) (Fig.10).These structures are governed by many factors including solvation, sterics, hydrogen bonding, stereo centers, and benzyl substitution patterns ( Scherrenberg et al)( Gorman and Smith).The interior is thus well-suited for encapsulation of guest molecules. The three parts of the dendrimer can be tailored specifically for the desired purposes, e.g. as dendritic sensors, drug vehicles or drugs. The multivalent surfaces on a higher-generationdendrimer can contain a very high number of functional groups. This makes the dendritic surfaces and outer shell well-suited to host–guest interactions where the close proximity of a large number of species is important. As the chains growing from the core molecule become longer and more branched (in 4 and higher generations) dendrimers adopt a globular structure (Caminati et al) . Dendrimers become densely packed as they extend out to the periphery, whichforms a closed membrane-like structure. When a critical branched state is reached dendrimers cannot grow because of a lack of space. This is called the ‘starburst effect’(Fischer and Vögtle). The increasing branch density with generation is also believed to have striking effects on the structure of dendrimers. They are characterized by the presence of internal cavities and by a large number of reactive end groups (Fig.11).

4-Properties of dendrimers:

4.1-Characterization:

4.1.1-Size and size distribution:

The molar mass of the dendrimer can be predictedmathematically (Tomalia et al) :

where: Mc : is the molar mass of the core, Mm : the molar mass of the branched monomer, Mt : the molar mass of the terminal groups, nc : the core multiplicity, nm : the branch-juncture multiplicity, G : the generation number.The increase of the number of dendrimer terminal groups is consistent with the geometric progression:

The monodispersed nature of dendrimers, has been verified extensively by mass spectrometry, size-exclusion chromatography, gel electrophoresis, and electron microscopy (TEM) (Jackson et al). as illustrated by TEMs for a Gen 5–10 series of PAMAM dendrimers (Fig. 12).

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Additional techniques to further elucidate structure-property relationships of dendrimers include fluorescence quenching, luminescence, and electrochemistry. Dendrimers pertain both to the molecular chemistry world for their step by step controlled syntheses, and to the polymer world because of their repetitive structure made of monomers; thus they benefit from analytical techniques from both worlds.

4.1.2-Structural Elucidation:

The ultimate proof of molecular structure is often reached by the determination of a crystal structure, In the case of dendrimers, however, this technique is of little use. The polymeric nature of dendrimers generally leaves their solid-state structure without any long-range order. Even when some degree of structure is present for instance in the case of very dense-surfaced spherical dendrimers there is such a degree of disorder within the inner generations that a crystal structure cannot be determined. There are a few examples of crystalline dendrimers and powder and single crystal." X-ray studies of dendrimers", but these are confined to low generations and rigid, hindered molecules. Nuclear Magnetic Resonance (NMR) spectroscopy and mass spectrometry have both been invaluable techniques in the characterization of dendrimers. Indeed, 1H and 13 C NMR spectra of dendrimers can be surprisingly simple and contain a great deal of information about defects and impurities in their structures. The mass spectrometry of dendrimers has benefited from the soft ionization techniques developed for the study of large biomolecules. Other methods, familiar to polymer chemists, have been used extensively in the characterization of dendrimers. Electron microscopy has been of great use in the visualization of dendrimers, and their aggregates, and Gel Permeation Chromatography (GPC) has been used for the calculation of radii of gyration, hydrodynamic radii, and polydispersities. Low Angle Laser Light Scattering (LALLS), Small Angle Neutron (SANS), and X-ray (SAXS) Scattering techniques have met with limited use.( AN Shipway)

4.1.3-Characterisation of the Dendritic Microenvironment:

Many investigators have made use of functional probes in order to study dendritic microenvironments. These probes can either be attached covalently to the dendritic structure, like the photochemical, chiral, and solvatochromic moieties , or they can be introduced as guest species.Spectroscopic methods have also produced information about dendritic microenvironments. PAMAM dendrimers have been shown20 to have decreased 13 C relaxation times for internal generations, suggesting that these moieties are less mobile than the surface groups.Rotational-Echo Double Resonance (REDOR), solid-state NMR spectroscopy has been used to examine the shape of the Fréchet polyethers, and Electron Spin Resonance (ESR) spectroscopy of complexed PAMAMs have been examined. Computer modelling of dendrimers has been used extensively for the purposes of visualization and dynamics experiments.( AN Shipway) This is review of the main analytical techniques used for the characterization of the chemical composition, the morphology, the shape, and the homogeneity of dendrimers. It includes NMR, IR, Raman, UV–Visible, fluorescence, circular

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dichroism, X-ray diffraction, mass spectrometry, SAXS, SANS, Laser Light Scattering, microscopy, SEC, EPR, electrochemistry, electrophoresis, intrinsic viscosity, DSC, and dielectric spectroscopy.

4.1.4-Analytical methods:

A-Size Exclusion Chromatography (SEC): Analysis was performed using an Alliance Waters 2690 separation module equipped with a Waters UV-Vis detector, a Wyatt Dawn laser photometer, Optilab interferometric refractometer and Waters ultrahydrogel columns. Phosphate buffer (0.05 M, pH 2.5) with 0.025% sodium azide was used as mobile phase. The flow rate was maintained at 0.6 mL/min. Sample concentration was kept at 2 mg/mL and 100μL was injected. Data was elaborated using Astra and PeakFit software.

B-Potentiometry:

Dendrimers were dissolved in NaCl (0.1 M) solution at concentration of 0.5 mg/mL, pH was set to around 3. In case of each sample the same number of moles (adjustedby the volume) was titrated. Titrations were performed at room temperature, under nitrogen atmosphere, using Molspin automated titration system, Mettler-Toledo combination In Lab electrode and NaOH (0.1 M) as a titrant.

C-Gel electrophoresis:

Dendrimers was performed using a vertical electrophoresis system (Model FB-VE10-1 Fisher Biotech) with a commercial power supply (Model EC135-90; Thermo Electron Corporation).

D-Matrix-assisted laser desorption ionization-time of flight mass spectrometry:

Spectra of dendrimer nanodevices were recorded on a Bruker Biflex IV spectrophotometer, using 2,5-dihydroxybenzoic acid (DHB) as a matrix. First 0. 5 μL of matrix was placed on target plated, and evaporated. Then 0.5 μL of the dendrimer (~1.5 mg/mL) was spotted over the matrix. Matrix and dendrimer were dissolved in 75% ACN and 0.1% TFA aqueous solution. For each spectrum 100 shots and 50% of laser power was applied.

E-Nuclear Magnetic Resonance:

Spectra were recorded using Bruker AMX 400 MHz, D2O as solvent and at sample concentrations 20 mg/mL.( Wojciech et al).

4.2-Physical and chemical properties:

Dendrimers are monodisperse macromolecules, unlike linear polymers. The classical polymerization process which results in linear polymers is usually random in nature and produces molecules of different sizes, whereas size and molecular mass of dendrimers can be specifically controlled during synthesis.

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In general, convergent methods produce the most nearly monodisperse dendrimers as determined by mass spectrometry. This is because the convergent growth process allows purification at each step of the synthesis and eliminates cumulative effects due to failed couplings (Fre´chet et al). Because of their molecular architecture, dendrimers show some significantly improved physical and chemical properties when compared to traditional linear polymers. Dendrimers are of interest for their unusual physical properties. Much work has been carried out in areas such as melt viscosity, glass transition temperature, rheological properties and even on the photo induced electron transfer to C60.The most exciting physical property of dendrimers is the variation of their intrinsic viscosities with molecular weight. It is found that, when the generation increases beyond a certain point, the intrinsic viscosity begins to decline, contrary to the behavior of linear polymers (Curve. 1). This effect is believed to be a consequence of the globular shapes of high generation dendrimers leaving them unable to 'tangle' with one another after the manner of linear polymers. ( AN Shipway)

In solution, linear chains exist as flexible coils, in contrast, dendrimers form a tightly packed ball. This has a great impact on their rheological properties. Dendrimer solutions have significantly lower viscosity than linear polymers (Fréchet). When the molecular mass of dendrimers increases, their intrinsic viscosity goes through a maximum at the fourth generation and then begins to decline (Mourey et al). Such behaviour is unlike that of linear polymers. For classical polymers the intrinsic viscosity increases continuously with molecular mass.The presence of many chain-ends is responsible for high solubility and miscibility and for high reactivity (Fréchet). Dendrimers’ solubility is strongly influenced by the nature of surface groups.Dendrimers terminated in hydrophilic groups are soluble in polar solvents, while dendrimers having hydrophobic end groups are soluble in nonpolar solvents.Dendrimers have some unique properties because of their globular shape and the presence of internal cavities. The most important one is the possibility to encapsulate guest molecules in the macromolecule interior. The observation of photo physical changes were the first evidence of encapsulation behavior of dendrimers(Hawker et al)( Fréchet et al). Hawker et al. coupled Frechet-type dendrons to a 4-(N-methylamino)-1- nitrobenzene core and observed an increase in the solvatochromic shift in the absorption spectra as the dendrimer generation increased (Hawker et al). The results indicated the larger generation dendrimers effectively shielded solvent and created an intrinsic microenvironment around the core. Photochemical modifications of the dendritic surface cause encapsulation and release of guest molecules. A fourth generation polypropylene imine dendrimer with 32 end groups was terminated in azobenzene groups (Fig.13). The azobenzene groups undergo a fully reversible photoisomerization reaction. The E isomer is switched to the Z form by 313 nm light and can be converted back to the E form by irradiation with 254 nm light or by heating. Such dendrimers can play the role of photoswitchable hosts for eosin Y. Dendrimers are attractive candidate molecules for studying electron transfer based solely on the intrinsic ability of the dendrons (for a given molecular weight) to effectively shield the core with organic material. The distance of electron travel and the material surrounding the core governs electron transfer rate (Gorman et al) (Fig.14) . This distance can be altered by 1) increasing the bulk material of the dendrons around the redox-active core, and/or 2) alter the dendritic architecture to create a dynamic conformational change of the dendrimer. Both strategies would result in an increase in

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the effective encapsulation and, subsequently, the distance of electron transfer to the redox active core. Already early in the history of dendrimers it was suggested that the 3-dimensional nanosized structure of the higher generation dendrimers would make this class of synthetic molecules suitable as mimics of proteins (Farin and Avnir).however, that in contrast to proteins which consist of folded, linear polypeptide chains, the branched architecture of the dendrimer interior is to a large extent formed by covalent bonds, resulting in a somewhat less flexible structure. In addition, the dendrimer is on average less compact than a protein, i.e. interior is not packed as efficiently as in typical proteins, and the dendrimer contains a substantially highernumber of surface functional groups than proteins of comparable molecular weight. (Table 1).Molecular dynamic studies carried out by several research groups on dendrimers show that the dendrimers, similar to proteins, can adapt ‘native’ (e.g. more tight) or ‘denaturated’ (e.g. extended) conformations dependent on the polarity, ionic strength and pH of the solvent.Amino-terminated PPI and PAMAM dendrimers (that is dendrimers having primary amines as surface groups) exhibit extended conformations upon lowering of pH because electrostatic repulsion between the protonated tertiary amines in the interior as well as between the primary amines at the dendrimer surface, forces the dendrimer branches apart (I. Lee et al) (Fig.15). At pH > 9 back-folding occurs as a consequence of hydrogen bonding between the interior protonated tertiary amines and the primary surface amines, resulting in a denser core (W. Chen et al).The pH-related conformational changes are dependent on the nature of the charged group at the dendrimer surface.For PPI dendrimers having surface carboxylic groups ‘small angle neutron scattering’ (SANS) and NMR measurements of the diffusion coefficients in aqueous buffer, showthat these dendrimers have the most extended conformations at pH 4 and pH 11, (Fig.15) This may be due to electrostatic repulsion between the protonated cationic inner tertiary amines at low pH, and electrostatic repulsion between the negatively charged deprotonated carboxylates at the dendrimer surface at high pH forcing the dendritic branches apart.At pH 6 the carboxy-terminated PPI dendrimer has no net charge, resulting in a tighter conformation controlled by intramolecular hydrogen bonding (W. Chen et al). Molecular density measurements at this pH show a homogeneous molecular density over the whole dendrimer, indicating a substantial degree of back-folding i.e. hydrogen bonding between terminal groups and groups in the core region (Fig.16).The polarity of the solvent greatly influences the 3-dimensional structure of dendrimers, Initial theoretical studies by de Gennes and Hervet on unmodified PAMAM (Starburst™) dendrimers using a self consistent mean-field model, concluded that in good solvents (that is solvents with a high ability to solvate the dendritic structure), the dendrimers had the highest molecular density at the periphery,leading to dense packing of the surface groups upon increasing generation (Gennes and

Hervet). Recent NMR studies performed on PPI dendrimers indicate that an a polar solvent such as benzene will favour polar intramolecular interactions (e.g. hydrogen bonding) resulting in back-folding of the dendrimer arms into the dendrimer interior, whereas the increased acidity of chloroform, will increase solvation of the dendrimeric structure via hydrogen bond donation to the interior tertiary amines resulting in a more extended conformation of the dendrimer (M. Chai et al). Both theoretical as well as experimental studies on amino functionalized PPI and PAMAM dendrimers reflect

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the tendency of an a polar solvent (poor solvent) to induce a higher molecular density in the core region due to back-folding (intramolecular polar interactions), and lower molecular density at the surface. In polar solvents the dendrimer arms are solvated and the molecular density at the dendrimer surface is increased (M. Ballauff). A microenvironment can arise in the dendrimer core as a consequence of limited diffusion of solvent molecules into the dendrimer. As an example, dendrimers dissolved in polar solvents such as aqueous media can have a very a polar interior (unimolecular micelle) allowing organic molecules to be encapsulated and carried in aqueous media. This property of dendrimers makes this class of molecules very well-suited as carriers of various bioactive substances.

4.3-Properties of dendrimers in biological systems:

Biological properties of dendrimers are crucial because of the growing interest in using them in biomedical applications. “Cationic” dendrimers (e.g., amine terminated PAMAM and poly(propyleneimine) dendrimers that form cationic groups at low pH) are generally haemolytic and cytotoxic.Their toxicity is generation-dependent and increases with the number of surface groups (Roberts et al).PAMAM dendrimers (generation 2, 3 and 4) interact with erythrocyte membrane proteins causing changes in protein conformation. These changes increase with generation number and the concentration of dendrimers. Anionic dendrimers, bearing a carboxylate surface, are not cytotoxic over a broad concentration range (Malik et al).

4.3.1-The significance of multivalency in biological interactions:

Multivalent interactions can be found throughout nature, ranging from the divalent binding of antibodies and many biological receptors to the multimillion-valent interactions of a Gecko’s foot hair (K. Autumn et al). Multivalency has been shown to lead to a strongly increased activity compared to the corresponding monomeric interaction.This synergistic enhancement of a certain activity e.g. catalytic activity or binding affinity from a monomeric to a multimeric system, is generally referred to as the ‘cluster’- or ‘dendritic’ effect ( W. B. Turnbull and J. F. Stoddart),( P. H. Ehrlich ),( A. F. Habeeb ),( J. Lundquist

et al). The dendritic effect is attributed to a co-operative effect in a multivalent system leading to a larger increase in activity than expected from the valency of the system (i.e. additive increase). The multivalent interactions provide:–tight binding from rather low-affinity binding of single ligands,–a possibility of utilizing low-affinity ligands in a new arrangement to cope with an evolutionary new binding partner,–more efficient cell-cell interactions mediated by multiple interactions.Factors that play a role in the binding of dendrimeric multivalent ligands include obviously the geometry of the multimerically presented ligands and the flexibility of their attachment to e.g. a dendrimer,(Fig. 17) (J. B. Corbell et al).

4.3.2-Biocompatibility of dendrimers:

In order to apply dendrimers as tools for drug design or as drug delivery devices in vivo, they have to fulfill several biological demands of crucial importance.

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The dendrimers should be:– non-toxic;– non-immunogenic (if not required e.g. for vaccines),– able to cross biobarriers such as, intestine, blood-tissue barriers, cell membranes, – able to stay in circulation for the time needed to have a clinical effect,– able to target to specific structures.

5-Applications:

5.1-Physical association and encapsulation of drugs:

Active pharmaceutical ingredients (APIs) can physically interact with dendrimers through either encapsulation into void spaces (nanoscale container) or association with surface groups (nano-scaffolding) or a mixture of both. Driving forces for these interactions are hydrogen bonding, van der Waals interactions, and electrostatic attraction between opposite charges on dendrimers and APIs. Small organic molecule drugs often are encapsulated into the dendrimers’ interior void space, while larger (bio)molecules preferably adsorb onto the dendrimer surface. The following listing of drugs physically associated with dendrimers provides an overview of the breadth of the dendritic platform to serve as drug carriers: Camptothecin, Cisplatin, Paclitaxel, Diclofenac and mefenamic acid, Doxorubicin, Etoposide, 5-Fluorouracil, Ibuprofen, Indomethacin, Ketoprofen, and others. Research activities are centered on three main classes of drugs: anticancer, anti-inflammatory, and antimicrobial drugs,(Fig.18).

5.2-Chemical conjugation of drugs to dendrimers (prodrug approach):

Physical interactions between dendrimers and APIs have specific features:

1. They leave the APIs unaltered, and therefore, provide a less challenging regulatory path forward.

2. They are easy to establish but provide limited control over release kinetics.3. They often allow only limited drug loading, resulting in rather poor drug-to-

dendrimer ratios.Alternatively, drugs can be conjugated to dendrimers via chemical bonding.

There are three main pathways to create these prodrugs:

1. Direct conjugation of drugs to the dendrimer surface.2. Conjugation via a linker molecule if the drugs do not carry the desired

functional group for direct conjugation or if the linker molecules are needed to modify solubility profiles or release kinetics, or reduce congestion of drug molecules on the dendrimer surface, allowing a higher degree of conjugation.

3. Drug molecules can become an integral part of the dendritic carrier that is released through certain triggering events at the desired location, e.g., a tumor site.

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Added benefits of the prodrug approach consist in modified pharmacokinetics and pharmacodynamics.

5.3-Drug delivery properties:

Dendrimers, which are capable of interacting specifically with cancerous tumor tissue, are an excellent option as a drug transporter within the body. Various polymers and other highly branched structures lack consistency of functional groups, monodispersive nature, and uniform molecular weight distribution. Dendrimers also have an exceptionally high drug loading capacity, which provides a greater accumulation of drug at the tumor site. Considering that dendrimers can be prepared with a predetermined, specific number of monomers and polymer branches, as well as peripheral functional group specific, they are the ideal macromolecule to enter the highly permeable vasculature of tumor sites and remain localized at the site to deliver an immense amount (within cytotoxic levels) of drug to the specific tissue . The surface of dendrimers can be modified with functional groups so that drugs will be physically entrapped, encapsulated, or conjugated by covalent bonds, ionic interactions, or hydrogen bonds. With the increase of molecular weight of the drug,due to the dendrimer-drug interaction, the hydrodynamic volume increases causinglonger circulation time and slower elimination of drug so cytotoxicity levels are lowered and dosage can be decreased. Lastly, dendrimers have the ability to solubilize some insoluble anti-cancer drugs. Increased solubility results in higher loading efficiency, which prevents nonspecific interactions and negative side effects of the drug.{1}

5.4-Dendrimers as drug delivery devices:

The research in dendrimer mediated drug delivery has mainly been focused on the delivery of DNA drugs (genes or gene inhibitors) into the cell nucleus for gene or anti-sense therapy, and numerous reports have been published on the possible use ofunmodified amino-terminated PAMAM or PPI dendrimers as non-viral gene transfer agents, enhancing the transfection of DNA into the cell nucleus.It has been found that partially degraded (or fragmented) dendrimers are better suited for gene delivery than the complete dendrimers , and a fragmentation (activation) step consisting of hydrolytic cleavage of the amide bonds is needed to enhance the transfection efficiency.In comparison to the intact dendrimers, the partially degraded dendrimers have a more flexible structure (fewer amide bonds) and form a more compact complex with DNA, which is preferable for gene delivery by the endocytotic pathway.The unmodified amino-terminated dendrimers transport the DNA to the cell membrane and may help in the transfection process by disruption of the cell membrane. The transfection of free DNA will be hampered by electrostatic repulsion between the negatively charged phosphate groups in the DNA backbone and the negatively charged cellular membrane.Tumors, obtain specific properties (increased vasodilatation, vasculature, etc.), can be combated with a new drug delivery system. The use of monodispersive, polymeric branched dendrimers as an anti-cancer drug delivery system has shown many promising results. Dendrimers can be developed with specific properties, which allow

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for targeting and releasing drugs at the target tumor site. This ultimately leads to lower dose rates, lower cytotoxicity levels, and higher drug efficiency.{2}

5.5-Folate conjugated dendrimers:

Dendrimers can be conjugated with a ligand specific for targeting tumor sites. Ligands can be recognized by the appropriate cell surface receptor and thus internalized where it can deposit the therapeutic agent. One such ligand being tested for use is folate. Folate appears to be an excellent ligand because many cancerous tumor cells over express folate receptors , As the folate receptor is over-expressed in cancer cells, these folic acid derivatised dendrimers are taken up by cancer cells preferentially to normal cells, making these dendrimers well-suited for the cancer specific drug delivery of cytotoxic substances. Very recently, folate modified PAMAM dendrimers have been successfully used as carriers of boron isotopes (10B) in boron neutron-capture treatment of cancer tumors.Studies have shown that by using the folate conjugated dendrimer encapsulated with methotrexate (antifolate drug), there has been better tumor targeting potential, which decreases tumors to a greater degree compared to the free drug.{1}

5.6-Vectors, in gene therapy:

Dendrimers can act as carriers, called vectors, vectors transfer genes through the cell membrane into the nucleus. Currently liposomes and genetically engineered viruses have been mainly used for this. PAMAM dendrimers have also been tested as genetic material carriers. They are terminated in amino groups which interact with phosphate groups of nucleic acids. This ensures consistent formation of transfection complexes. A transfection reagent called "SuperFect" consisting of activated dendrimers is commercially available. Activated dendrimers can carry a larger amount of genetic material than viruses. SuperFect–DNA complexes are characterized by high stability and provide more efficient transport of DNA into the nucleus than liposomes. The high transfection efficiency of dendrimers may not only be due to their well-defined shape but may also be caused by the low pK of the amines (3.9 and 6.9). The low pKpermit the dendrimer to buffer the pH change in the endosomal compartment.{3}

5.7-Glycodendrimers:

Glycodendrimers find use as a targeted anticancer drug vehicle because of the relationship of glycosylation on cancer cell surfaces. This leads to the over expression of specific antigens that serve as targets for immune recognition by interacting with lectin-like receptors present on immune cells. Glycodendrimers can be used in a similar manner by being introduced to the tumor site and acting as an antigen to promote the accumulation of monoclonal antibodies, which then selectively affect tumor cells. Glycodendrimers closely resemble the natural carbohydrate ligands, which add to the selective localization of the drug to the specific site. As with other dendrimers, the drug is circulated longer and delivered with greater efficiency. The formation of these dendrimers can occur in three ways:

Fully coated dendrimers with carbohydrates, Carbohydrate moieties at the periphery, and Carbohydrates at the center of the dendrimer.{1}

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5.8-PEGylated dendrimers:

PEG modification on the surface of the dendrimers is receiving more attentionsbecause it increases water solubility and biocompatibility of the dendrimers fordrug delivery.{4}

Polyethylene glycol (PEG) dendrimers are non-toxic and avoid detection by the immune system, protein adsorption, and show tissue specificity. By inserting drugs into PEGylated dendrimers, there is an improved efficiency of delivery to the tumor site of that drug and an increase in the half-life because of the prevention of deterioration due to non-specific interactions. This increase in stability ultimately results in the reduction of doses, increased tumor selective uptake, and reduced allergic responses. Using PEGylated dendrimers also has the advantage of reduced cytotoxicity and immunogenicity.A drawback in other dendrimer system. Kojima et al. reported that PEG solubility is affected by varying the chain length. Studies were done with varying chain sizes and by encapsulating anti-cancer drugs, methotrexate, and doxorubicin. The results of the study showed that with an increase chain length, there was an increase in encapsulation efficiency and dendrimer generation was seen.{1}

5.9-Peptide dendrimers:

Peptide dendrimers consist of amino acids as the core molecules with the outer polymers made up of peptide moieties or amino acids. These dendrimers are effective as drug vehicles because peptides can trigger apoptosis and inhibit the growth of epithelial tissues. A common type of peptide dendrimer is the L-lysine dendrimer, which has shown to be a molecular inhibitor of angiogenic factors. Also, the L-lysine dendrimer has shown to stimulate an immune response creating antibodies specific against tumors. Other methods in which peptides act in a medicinal way are the obstruction of cell cell interactions and the prevention of cell adhesion.{1}

5.10-PH sensitive dendrimers:

Dendrimers of this type generated to exhibit specific properties which allow them to be small enough to enter normal vessels, as well as being of optimal size to enter thetumor through the increased pore size. Once inside the cell or tumor site there is a change in pH, which then affects the conformation of the dendrimer . One method of how pH sensitive dendrimers are used for drug transport is that at normal physiological pH (7.4) the terminal amine groups on a polypropylene imine dendrimerare not protonated and the branches converge onto the center multifunctional molecule or drug. Upon entering the microenvironment of the tumor site, pH drops to a more acidic level and the terminal amine groups are now protonated and repel one another. This stimulates the release of the drug at the tumor site by the opening of the branches outward. pH sensitive dendrimers can also be fitted with functional groups on the surface of the dendrimer. By adding various functional groups such as an amine to the dendrimer surface, release of drugs occurs when the functional group protonates. Additionally, because the release of the drug is pH dependent, the degree of side effects and cytotoxicity decreases.{1}

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5.11-Dendrimers in Topical Drug Delivery:

Surface modifications of dendrimers have been used as molecular-carrying systems. For example, a keratolytic or anti-acne agent was complexed with a carrying molecule such as a dendrimer containing free amino groups to obtain cosmetically acceptable formulations for treatment of acne vulgaris. In another example of a dendrimer-molecule conjugate system, coupling of amino butadiene with an amine-rich dendritic molecule provided advantageous UV-absorbing capabilities to the final product. This high-molecular-weight dendrimer-amino butadiene-complexed molecule allowed ease in formulating a clear sunscreen composition without developing high-viscosity gels, which in turn provided ease of application to the skin. Because of the high molecular weight of the resulting molecule, it was nonpenetrating into the skin, which would minimize risk of irritation or sensitization reactions while acting as a UV-light absorber when applied on the skin's surface. In another application, amine-terminated cationic dendrimers have been used in personal-care cleansing compositions as mildness agents. Linear cationic polymers used as mildness agents usually precipitate in the presence of anionic surfactants, which reduces their lathering, skin conditioning, or cleansing effects. Dendrimers, on the other hand, are capable of interacting favorably and can bind with anionic surfactants in the composition to remain dispersed in salt solutions. This interaction of cationic dendrimers with skin-irritating anionic surfactants could potentially be used for reducing the skin irritation potential of cosmetic formulations containing harsh anionic surfactants.{5}

5.12-Dendrimers in transdermal delivery:

Dendrimers have found recent applications in novel transdermal delivery system, providing benefits such as improved drug solubilization, controlled release, and drug-polymer conjugates (pro-drugs). The viscosity-generation-number property of a dendrimer solution allows for ease of handling of highly concentrated dendrimer formulations for these applications. Dendrimers have been shown to be useful as transdermal drug delivery system for nonsteroidal anti-inflammatory drugs (NSAIDs), antiviral, antimicrobial, anticancer, or antihypertensive drugs. PAMAM dendrimers have been studied as carrier transdermal systems for the model NSAIDs: ketoprofen and diflunisal. It was found that the PAMAM dendrimer-drug formulations showed increased transdermal drug delivery compared with formulations lacking dendrimers. In vivo studies in mice showed prolonged pharmacodynamic responses and 2.73-fold higher bioavailability over 24 h for certain dendrimer-containing drug solutions.{5}

5.13-Dendrimers as ophthalmic vehicles:

The majority of topically applied ocular drug-delivery systems are formulated either as solutions, ointments, or suspensions and suffer from various disadvantages such as quick elimination from the precorneal region, poor bioavailability, or failure to deliver the drug in a sustained fashion. Several research advances have been made in ocular drug delivery systems by using specialized delivery systems such as polymers, liposomes, or dendrimers to overcome some of these disadvantages. Ideal ocular drug-delivery systems should be nonirritating, sterile, isotonic, biocompatible, and biodegradable. The viscosity of the final product should be optimized so that the dosage form does not run out of the eye. Dendrimers provide solutions to some complex delivery problems for ocular drug delivery.

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In particular, the cationic dendrimers are of interest for their application in the ocular topical pathway since they exhibit high interaction with the mucins of the corneal epithelium which are loaded negatively in physiological conditions by the presence of sialic groups. This electrostatic interaction converts these dendrimeres in mucoadhesive compounds able to generate an increase in the contact period of the pharmaceutical form (and the drug that it contains) on the surface of the eye. In addition, the cationic nature of these dendrimeres also induces electrostatic interaction with proteins of the epithelial intercellular unions, generating a temporary reorganization of these structures and an increase of their para cellular permeability. These properties are attractive for the use of these polymers as promotional agents of the penetration of active substances through the cornea.{6} In the New Zealand albino rabbit model, the residence time of pilocarpine in the eye was increased by using dendrimers with carboxylic or hydroxyl surface groups. These surface-modified dendrimers were predicted to enhance pilocarpine bioavailability.{5}

5.14-Dendrimer drugs:

A-Dendrimers as antiviral drugs:

In general, antiviral dendrimers work as artificial mimics of the anionic cell surfaces, thus the dendrimers are generally designed having anionic surface groups such as sulfonate residues or sialic acid residues, which are acidic carbohydrates present at themammalian cell surface. In other words, the dendritic drug competes with the cellular surface for binding of virus, leading to a lower cell-virus infection probability.Polylysine dendrimers modified with naphtyl residues and having sulfonate surface groups have been found to be useful as viral inhibitors for Herpes Simplex virus in vitro. PAMAM dendrimers covalently modified with naphthyl sulfonate residues at the surface, giving an polyanionic surface, also show antiviral activity against HIV. Also here the dendrimer drug works as an inhibitor for early stage virus/cell adsorption and at later stages of viral replication by interfering with the reverse transcriptase and/or integrase enzymes.{7}

B-Dendrimers as antibacterial drugs:

In contrast to the antiviral dendrimers, the antibacterial dendrimers generally contain cationic surface functionalities such as amines or tetra alkyl ammonium groups. The general mode of action of the antibacterial dendrimer is to adhere to and damage theanionic bacterial membrane, causing bacterial lysis.PPI dendrimers where the surface has been functionalized with tertiary alkyl ammonium groups have shown to be very potent antibacterial biocides against Gram positive and Gram negative bacteria.Polylysine dendrimers having mannosyl surface groups have been shown to inhibit adhesion of E. coli to horse blood cells in a haemagglutination assay, making these structures promising as antibacterial agents.{7}

5.15-Dendrimers as protein denaturants:

Certain types of dendrimers act as chaotropes i.e. water structure perturbing solutes, lowering the dielectric constant and the viscosity of water; disordering the regular water structure by reorganizing water molecules at the dendrimer surface. As with

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other chaotropes, this leads to hydrophobic interactions being disfavored which, in turn, is highly destabilizing for most protein tertiary structures (denaturation).Classical examples of chaotrophic salts are MgCl2, urea, guanidinium chloride, sodium thiocyanate, guanidinium thiocyanate at high concentrations and other chaotropes include polarity-decreasing, water miscible organic solvents such as acetonitrile, propanol and methanol. Generally, chaotropes will serve to denature and solubilise proteins, which is useful for example in solubilising protein aggregates as are often encountered when expressing proteins in heterologous expression systems (inclusion body formation) and when extracting certain types of membrane proteins. Dendrimers, being compact, large polyionic substances have the physicochemical properties needed to make them potential chaotropes/protein denaturants.A striking example of this was cationic dendrimers were used for the solubilisation of prion protein aggregates. Prion proteins are able to attain a pathogenic structure/conformation in which they can cause mortal diseases called spongiform encephalopathy's, including mad cow disease and Creutzfeldt-Jakob disease. These deadly conformers are characterized by their tendency to form very insoluble aggregates, which are found in the brains of affected individuals. Such aggregates are soluble only in solvents containing both detergent and denaturant (typically 6 M guanidinium chloride), however it was shown that such aggregates can be solubilised by cationic dendrimers, such as PEI-, PPI- and PAMAM dendrimers, higher generation ( > G3) dendrimers being the most efficient and influenced by the number of surface amino groups. PAMAM dendrimers having hydroxyl groups at their surface (PAMAM-OH)and linear polymers had no or very minor effects. The effect was seen at surprisingly low concentrations (7 mg ml21 or below) on aggregate producing neuroblastoma cells and took place with no cytotoxicity.{7}

5.16-Dendrimers in vaccines:

It is well-established that small molecular weight substances (e.g. peptides) are not very immunogenic. i.e. no or a weak immune response (including antibody formation) is induced upon their injection into a recipient host. However, this problem can be overcome by increasing the molecular weight of the substance in question either by polymerization or by coupling it to a multifunctional, high molecular weight carrier, traditionally a naturally derived protein. For the preparation of highly defined, reproducible immunogens e.g. for human vaccine uses, other types of carriers are highly desirable and in this respect, dendrimers have emerged as useful since they can act as multivalent and well defined carriers for antigenic substances by coupling of antigen molecules to the surface functional groups of the dendrimer.Examples of dendrimer–peptide compounds being used for vaccine and immunization purposes include the multiple antigenic peptide (MAP) dendrimer system pioneered by Tam and coworkers, which can be synthesized with defined mixtures of B andT-cell epitopes, either synthesized by stepwise peptide synthesis on the branches of the MAP or by segment coupling of peptide fragments by various methods.

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6-Conclusion:

Dendritic polymers are expected to play a key role as enabling building blocks for nanotechnology during the 21st century. The controlled shape, size, and differentiated functionality of dendrimers, their ability to provide both isotropic and anisotropic assemblies, their compatibility with many other nanoscale building blocks such as DNA, metal nanocrystals, and nanotubes, their potential for ordered self-assembly, their ability to combine both organic and inorganic components, and their propensity to either encapsulate or be engineered into unimolecular functional devices make dendrimers uniquely versatile amongst existing nanoscale building blocks and materials.Dendritic polymers, especially dendrons and dendrimers, are expected to fulfill an important role as fundamental modules for nanoscale synthesis. It is from this perspective that it is appropriate to be optimistic about the future of this new major polymer class, the dendritic state.

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